Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation

Biomarker candidates of neurodegeneration in Parkinson’s disease for the evaluation of disease-modifying therapeutics

Reliable biomarkers that can be used for early diagnosis and tracking disease progression are the cornerstone of the development of disease-modifying treatments for Parkinson’s disease (PD). The German Society of Experimental and Clinical Neurotherapeutics (GESENT) has convened a Working Group to review the current status of proposed biomarkers of neurodegeneration according to the following criteria and to develop a consensus statement on biomarker candidates for evaluation of disease-modifying therapeutics in PD. The criteria proposed are that the biomarker should be linked to fundamental features of PD neuropathology and mechanisms underlying neurodegeneration in PD, should be correlated to disease progression assessed by clinical rating scales, should monitor the actual disease status, should be pre-clinically validated, and confirmed by at least two independent studies conducted by qualified investigators with the results published in peer-reviewed journals. To date, available data have not yet revealed one reliable biomarker to detect early neurodegeneration in PD and to detect and monitor effects of drug candidates on the disease process, but some promising biomarker candidates, such as antibodies against neuromelanin, pathological forms of α-synuclein, DJ-1, and patterns of gene expression, metabolomic and protein profiling exist. Almost all of the biomarker candidates were not investigated in relation to effects of treatment, validated in experimental models of PD and confirmed in independent studies

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Time to reconfigure balancing behaviour in man: changing visual condition while riding a continuously moving platform.

While balancing on a continuously anteroposterior (A-P) translating platform (10 cm, 0.5 Hz), the head normally oscillates with the platform without vision but is stabilized in space with vision. We estimated the time to shift from one to the other balancing behaviour when visual condition changed at some stage during the balancing trials. Ten subjects performed randomly 50 balancing trials (each lasting 18 s): 10 trials with eyes open (EO), 10 with eyes closed (EC), 15 in which participants started with EO and closed their eyes (condition EO!EC) in response to an acoustic signal delivered during the trial, and 15 starting with EC and closing their eyes (EC!EO) in response to the same signal. No other speciWc instruction was given. Displacements of malleolus, hip and head, and EMG from leg and axial muscles were recorded. Indexes of amplitude of A-P head and hip oscillation and of amplitude of EMG activity were computed. All variables were larger with EC than EO. On changing visual condition during the trial, the pattern of head and hip movement and of muscle activity turned into that appropriate for the new visual condition in a time-interval ranging from about 1 to 2.5 s. For each subject, the mean latency of the change in the balancing behaviour was assessed by statistical methods. On average, the latencies of kinematics and EMG changes proved to be longer for the EO!EC condition than viceversa. Further, the latencies of the changes were also measured across all EO!EC and EC!EO individual trials. These values were clustered around particular epochs of the Wrst few oscillation cycles following the shift in visual condition. The results show that subjects can rapidly adapt their balancing behaviour to the new visual condition. However, they appear to refrain from releasing the new behaviour were this unWt, and unfastened it at appropriate time in the next platform translation cycle. These Wndings reveal the temporal and spatial features of the automatic release of the new balancing strategy in response to a shift in the ongoing sensory set, and emphasize the swiftness in the change in balancing behaviour when subjects pass from a non-visual to a visual reference frame

The control of equilibrium in Parkinson's disease patients: delayed adaptation of balancing strategy to shifts in sensory set during a dynamic task

Processing of sensory information, timing operations and set-shifting can be affected in Parkinson's disease (PD) patients. We investigated their capacity and swiftness to pass from a kinaesthetic- to a vision-dependent behaviour during dynamic balancing on a continuously moving support base. Nineteen on-phase PD patients and 13 age-matched normal subjects stood on a platform continuously translating in the antero-posterior direction at 0.2 Hz. Body segment oscillations were identified by a stereophotogrammetric device and electromyogram (EMG) was recorded from tibialis anterior and soleus. Under constant visual conditions, both patients and normal subjects roughly stabilised head and trunk in space with eyes open (EO) but followed the platform displacement with eyes closed (EC). Amplitude and variability of the periodic EMG bursts were smaller with EO than EC. Constant visual-condition trials were intermingled with trials in which subjects opened (EC-EO) or closed (EO-EC) the eyes in response to an acoustic signal. Both patients and normal subjects changed kinematics and EMG patterns to those appropriate for the new visual condition. However, PD patients were slower in changing their behaviour under the EC-EO condition. These findings show abnormal temporal features in balancing strategy adaptation when shifting from kinaesthetic to visual reference in PD. The delay in the implementation of the vision-dependent behaviour was unexpected, given the advantage vision is supposed to confer to motor performance in PD. This condition may play a major role in the instability of patients performing dynamic postural tasks under changing sensory conditions

Post-effect of forward and backward locomotion on body orientation in space during quiet stance

Neural circuits responsible for stance control serve other motor tasks as well. We investigated the eVect of prior locomotor tasks on stance, hypothesizing that postural post-eVects of walking are dependent on walking direction. Subjects walked forward (WF) and backward (WB) on a treadmill. Prior to and after walking they maintained quiet stance. Ground reaction forces and centre of foot pressure (CoP), ankle and hip angles, and trunk inclination were measured during locomotion and stance. In WF compared to WB, joint angle changes were reversed, trunk was more Xexed, and movement of CoP along the foot sole during the support phase of walking was opposite. During subsequent standing tasks, WB induced ankle extension, hip Xexion, trunk backward leaning; WF induced ankle Xexion and hip extension. The body CoP was displaced backward post-WB and forward post-WF. The post-eVects are walking-direction dependent, and possibly related to foot-sole stimulation pattern and trunk inclination during walking

Head stabilization on a continuously oscillating platform: the effect of a proprioceptive disturbance on the balancing strategy

When standing and balancing on a continuously and predictably moving platform, body equilibrium relies on both anticipatory control and proprioceptive feedback. We have vibrated different postural muscles of the body to assess any effect of confounding the proprioceptive input on balance during such unstable conditions. Low and high platform oscillation frequencies were used, because different strategies are used to withstand the two perturbations. Eyes open (EO) and closed (EC) conditions were also tested, to assess whether the stabilizing effect of vision is independent from the proprioceptive disturbance. Subjects (n = 14) performed two series of trials, EO and EC: (1) quiet erect stance, (2) stance on the platform translating at 0.2 or 0.6 Hz sinusoidally in the anteroposterior (A-P) direction (dynamic conditions). Continuous bilateral vibration (90 Hz) was produced by two vibrators fixed to the following homonymous muscles: dorsal neck, quadriceps, biceps femoris, tibialis anterior, and triceps surae. Acquisition of body segments' displacement began 10 s after the start of platform translation. From markers fixed to head, hip, and malleolus, we computed the A-P oscillation of head and hip, body orientation in space, and cross-correlation (CC) and time-delay between malleolus and head trajectories. The results were (a) the head A-P oscillation was smaller with EO than EC, under both quiet stance and dynamic conditions; (b) vibration of tibialis and triceps surae, but not of other muscles, slightly increased head and body A-P oscillation with EC under dynamic conditions; (c) at 0.2 Hz but not at 0.6 Hz, for all visual and vibration conditions, there was a significant association between head and feet; (d) at 0.2 Hz, EC, neck muscle vibration increased this association, whereas vibration of the other muscles induced a major time delay in the oscillation of head compared with feet; (e) vibration of either neck or tibialis induced forward body leaning, while vibration of either triceps surae or biceps femoris induced backward leaning, with both EO and EC, under both static and dynamic conditions; (f) the head A-P oscillation, however, under dynamic conditions was not dependent on body leaning. The relatively scarce effects of proprioceptive disturbance on head stabilization and multijoint coordination (in spite of effects on body orientation similar to those observed during stance) speak for a major role of anticipatory control in the dynamic equilibrium task. However, the significant vibration-induced time delay in segments' coordination at low translation frequency, EC, suggests that the normally patterned Ia input promotes continuous adjustments of the feed-forward control mode

Head stabilization on a continuously oscillating platform: the effect of a proprioceptive disturbance on the balancing strategy.

When standing and balancing on a continuously and predictably moving platform, body equilibrium relies on both anticipatory control and proprioceptive feedback. We have vibrated different postural muscles of the body to assess any effect of confounding the proprioceptive input on balance during such unstable conditions. Low and high platform oscillation frequencies were used, because different strategies are used to withstand the two perturbations. Eyes open (EO) and closed (EC) conditions were also tested, to assess whether the stabilizing effect of vision is independent from the proprioceptive disturbance. Subjects (n=14) performed two series of trials, EO and EC: (1) quiet erect stance, (2) stance on the platform translating at 0.2 or 0.6 Hz sinusoidally in the anteroposterior (A-P) direction (dynamic conditions). Continuous bilateral vibration (90 Hz) was produced by two vibrators fixed to the following homonymous muscles: dorsal neck, quadriceps, biceps femoris, tibialis anterior, and triceps surae. Acquisition of body segments’ displacement began 10 s after the start of platform translation. From markers fixed to head, hip, and malleolus, we computed the A-P oscillation of head and hip, body orientation in space, and cross-correlation (CC) and time-delay between malleolus and head trajectories. The results were (a) the head A-P oscillation was smaller with EO than EC, under both quiet stance and dynamic conditions; (b) vibration of tibialis and triceps surae, but not of other muscles, slightly increased head and body A-P oscillation with EC under dynamic conditions; (c) at 0.2 Hz but not at 0.6 Hz, for all visual and vibration conditions, there was a significant association between head and feet; (d) at 0.2 Hz, EC, neck muscle vibration increased this association, whereas vibration of the other muscles induced a major time delay in the oscillation of head compared with feet; (e) vibration of either neck or tibialis induced forward body leaning, while vibration of either triceps surae or biceps femoris induced backward leaning, with both EO and EC, under both static and dynamic conditions; (f) the head A-P oscillation, however, under dynamic conditions was not dependent on body leaning. The relatively scarce effects of proprioceptive disturbance on head stabilization and multijoint coordination (in spite of effects on body orientation similar to those observed during stance) speak for a major role of anticipatory control in the dynamic equilibrium task. However, the significant vibration-induced time delay in segments’ coordination at low translation frequency, EC, suggests that the normally patterned Ia input promotes continuous adjustments of the feed-forward control mode